Immunohistochemistry (IHC) and in situ hybridization (ISH) are in vitro laboratory procedures used to detect and diagnose abnormal conditions, such as cancer. IHC requires detecting proteins in cells of a tissue section by hybridizing antibodies to antigens, while ISH hybridizes radiolabelled complementary DNA/RNA probe to DNA/RNA within a tissue. The procedures are also widely used in basic research to understand the distribution and localization of biomarkers and differentially expressed proteins/DNA in different parts of a biological tissue.
According to the FDA Draft “Guidance for Industry Use of Histology in Biomarker Qualification Studies”, released December 2011, a biomarker is defined as: “a characteristic that is objectively measured and evaluated as an indicator of normal biologic processes, pathogenic processes, or biological responses to a therapeutic intervention”. The report goes on to state that a poorly characterized biomarker can do considerable harm. A poorly characterized biomarker may lead to inappropriate removal of a drug from development, encourage development of a drug that is unlikely to be approved, or lead to an erroneous perception of safety.
Positive and negative experimental controls for IHC and ISH are created from archived tissue blocks, or from cell lines embedded in tissue medium (i.e. formalin fixed paraffin embedded-FFPE). A positive control comprises a tissue or cell block containing the biomarker (antigen/DNA/RNA) of interest. Cell lines or purified cells from tissues or blood samples of affected patients are a valuable tool to provide known positive controls. These cells can provide a homogeneous tissue in a desirable amount, and properties can be controlled by molecular, cytogenetic and biochemical characteristics. Cells created in a paraffin block can provide sections for studying ex-vivo cell cycles, signaling pathways, and activation-dependent effects with biochemical agents. Cells grown in culture can be cytospun or prepared as cell smears on glass slides, however, conditions are quite different from tissue blocks which has been fixed, dehydrated and embedded in paraffin.
Cell pellets fixed in formalin become very tough, therefore embedding the pellet into paraffin creates a pellet with very dense cells. It is difficult to create cell blocks that serve as positive controls that are of a uniform density because of the clumping of the cells. And, without uniform density, cell block staining, image scanning and analysis may be inconsistent.
The current invention is to create a “homogeneous” cell block that has superior homogeneity of cells evenly distributed within the section to provide an even distribution of cells which will be used as a control standard.
The lack of reliable controls in IHC/ISH studies is a recognized problem. For example, it was recently noted that “ . . . IHC as a platform for biomarkers has been challenged by more quantitative molecular assays with reference standards but that lack morphologic context. For IHC to be considered a “top-tier” biomarker assay, it must provide truly quantitative data on par with non-morphologic assays, which means it needs to be run with reference standards . . . . Until pathologists can run immunohistochemically assays with known controls that serve as true reference standards, the use of MC on histologic sections will be considered a less valued or preliminary method of defining a biomarker” (Dunstan et al, Toxicologic Pathology, 39: 988-1002, 2011).
Therefore, there is a need within the medical and research industries to produce reliable controls, such as a cell block of uniformly distributed cells, or ratio of cells, for use as a positive control for a biomarker in immunohistochemistry slide scanning and image analysis.